带有CFP标签的LC3自噬表达载体的构建

目的构建带有樱桃红色荧光蛋白(cherry fluorescent protein,CFP)标签的微管相关蛋白1轻链3(LC3)自噬真核表达载体,并进行初步表达鉴定。方法采用PCR方法,从质粒载体pCMV-GFP-LC3为模板扩增LC3片段,通过T4 DNA连接酶将纯化后的PCR产物LC3与经过ApaI和BamHI双酶切的pm-cherry-c1载体连接,命名为PM-CFP-LC3。该载体经酶切及测序验证后,在fugene6转染试剂介导下转染293细胞,通过荧光显微镜观察CFP在细胞内的分布来观察LC3表达情况。结果带有CFP标签的LC3表达载体构建成功,在荧光显微镜下可观察到转染细胞中樱桃红色荧光以及细胞自噬现象。结论成功构建了带有CFP标签LC3的重组质粒并成功表达,为深入研究细胞的自噬奠定了实验基础。

Expression vector construction of LC3 autophagy with CFP tag

Objective To construct LC3 expression vector with CFP(cherry fluorescent protein) tag as well as verify the expression. Methods The LC3 sequence was amplified by PCR with pCMV-GFP-LC3 plasmid as template respec-tively. Purified PCR product LC3 was linked with revised pm-cherry-c1 vector digested by restriction enzymes ApaI and BamHI via T4 DNA ligase. The recombined plasmids named PM-CFP-LC3 and PM-CFP-LC3 were identified by sequencing, and then transfected into 293 cells by fugene6 oligfectamine reagent. The distribution of CFP in cells and the expression of LC3 was detected by fluorescence microscopy. Results The eukaryotic expression vector of LC3 with CFP tag was successfully constructed. The cherry fluorescence and the phenomenon of cell autophagy were detected by fluorescence microscopy. Conclusion Recombinant plasmid LC3 with CFP tag has been successfully constructed and expressed, which could be the basis of experiment in cell autophagy.